The present invention relates to oligopeptides derived from fragments of the C-reactive protein (hereinafter CRP), and to their use in the therapy of cardiovascular and inflammatory diseases.
CRP is a protein generally having a very low blood concentration, which rises up to two thousand times following inflammatory processes [J. J. Morley and I. Kushner, Am. N.Y. Acad. Sci., 389, 406-418 (1989)]. F. A. Robey et al., J. Biol. Chem., 262, No. 15, 7053-7057 (1987) disclose three CRP tetrapeptide sequences very similar to the ones of tuftsin. The chemically synthetized tetrapeptides stimulate the fagocytic leukocytes and the production of superoxide, and induce mononuclear cells to produce interleukin 1, in a tuftsin-like manner. Like tuftsin, the three CRP tetrapeptides are rapidly metabolized and inactivated by proteases in vivo.
It has been now surprisingly found that chemically modified analogues of said CRP tetrapeptide fragments are useful in the therapy of cardiovascular and inflammatory diseases, for example in the therapy of ischemia and/or ischemia and reperfusion state or the septic shock.
Therefore, the present invention relates to oligopeptides of formula (I)
A1xe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x83xe2x80x83(I)
wherein A1 is an aminoacid residue selected from the group consisting of threonine, leucine, isoleucine, valine, sarcosine, alanine, glycine and (C2-6)acyl-glycine, or is absent;
A2 is proline or N-xcex1-substituted basic aminoacid residue selected from the group consisting of lysine, arginine and ornithine, N-xcex1-substituted by at least one (C1-6)alkyl, benzyl or (C2-6)acyl group;
A3 is an aminoacid residue selected from the group consisting of proline, leucine, isoleucine and valine;
A4 is an aminoacid residue selected from the group consisting of arginine, leucine and glutamine, optionally amidated or esterified at the C-terminal position, or is an agmatine residue, or is absent;
when A2 is N-xcex1-substituted lysine, both A1 and A4 may be absent; the side-chain groups of the above aminoacid residues and of the agmatine residue may be optionally substituted by one or more (C1-6)-alkyl, benzyl or (C2-6)acyl groups, and each of said aminoacid residues may be in D- or L-form, or in form of one of the possible diastereoisomers or enantiomers; and their salts with pharmaceutically acceptable acids or bases.
A preferred group of compounds according to the invention are the ones of formula (I) wherein A1 is an aminoacid residue selected from the group consisting of glycine, threonine, leucine, isoleucine, valine, sarcosine, alanine (C2-6)acyl-glycine, or is absent;
A2 is lysine, arginine or ornithine N-xcex1-substituted by a (C1-6)alkyl, benzyl or (C2-6)acyl group; A3 is proline; A4 is glutamine, leucine, arginine, optionally amidated or esterified at the C-terminal position, or an agmatine residue, or is absent;
the side-chain groups of said aminoacid residue and of the agmatine residue may optionally be substituted by one or more substituents selected from the group consisting of (C1-6)alkyl, benzyl or (C2-6)acyl; and each of said aminoacid residue may be in D or L form, or in form of one of the possible diastereoisomers or enantiomers;
and the pharmaceutically acceptable acid or base salts thereof.
As (C1-6)alkyl it is intended a group such as methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert.-butyl, n-pentyl, 3-methyl-pentyl, n-hexyl group, and the relevant positional isomers. As (C2-6)acyl it is intended a group such as formyl, acetyl, propionyl, butyryl, pentanoyl, hexanoyl and the relevant positional isomers.
Another object of the present invention relates to the use of the oligopeptides of formula (I) as useful agents, in the therapy of cardiovascular and inflammatory diseases, such as those induced by ischemia and/or ischemia and reperfusion state and septic shock.
The compounds of the general formula (I) may be prepared employing peptide synthesis procedures, both in solid phase or in solution, known to the skilled in the art [see, for example, Merrifield, R. B., Biochemistry, 3, 1385 (1964)]. Unless otherwise mentioned, the aminoacid residues are intended to be used in L-configuration at the Cxcex1.
Preferably, the synthesis is carried out in solution starting from the selected aminoacid and assembling the oligopeptide by a step-by-step addition of the desired aminoacids. Anyway, pre-constituted di- or tripeptide units may also be employed. Even if the synthesis of the oligopeptide may be started from any aminoacid, and may proceed both in the N-terminal or C-terminal direction, it is preferable to carry it out in the N-terminal direction. The aminoacids or, if desired, the pre-constituted di- or tripeptides may be used as such or in form of the relevant derivatives protected at the carboxy group by esterification, e.g. with tert.-butyl (tBu) group, or/and at the amine group by amidation, e.g. with benzyloxycarbonyl (Z), and, in case, suitably protected at the side-chain groups, e.g. with 2,2,5,7,8-pentametyl-chroman-6-sulfonyl (Pmc), tert.-butyloxycarbonyl (Boc) or trifluoroacetic acid (TFA). These protections may be effected by methods familiar to the skilled in the peptide chemistry.
Anyway, the above mentioned protected derivatives are commercially available products too. The protective group of the xcex1-amino moiety is advantageously removed before the condensation with the subsequent aminoacid, for example through acidolysis with medium strong acids (e.g., trifluoroacetic acid), or through catalytic hydrogenolysis using gaseous hydrogen or hydrogen donors such as, for example, formic acid or salts thereof, triethylsylane, hydrazine in alkali, etc., selected in view of the aminoacid to deprotect and of the others, if present, in the presence of a suitable palladium catalyst. Then, the condensation with the subsequent aminoacid residue is carried out, such residue being suitably protected at the moieties not to be involved in said reaction.
Such condensation may be effected through one of the several known methods. Specifically, active esters may be used, e.g. succinimide (Su), fluoride (F), or condensing agents such as benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), bromo-tris-pirrolidium-phosphonium hexafluorophosphate (PyBroP), dicyclohexylcarbodiimide (DCC), etc., optionally in the presence of a catalyst such as 1-hydroxy-benzotriazole (HOBT), 4-dimethyl-amino-pyridine (DMAP), triethylamine (TEA), N-methyl-morpholine, N-methyl-imidazole etc.
As for the N xcex1-alkyl-derivatives of aminoacids, they are obtained by treatment with a suitable aldehyde, at low temperature, in the presence of cianoborohydride, selective reducing agent, in a polar solvent, preferably methanol.
When an oligopeptide of formula (I) having the C-terminal position in form of an amide is desired, commercially available aminoacids bearing such moiety may be employed as starting materials, or the C-terminal aminoacid may be amidated with an HOBT ammonium salt or, when A4 is an agmatine residue, with agmatine itself.
The compounds of formula (I) wherein A1 and/or A2 are acylated aminoacid residues, are obtained by treatment with a suitable acyl-anhydride, at low temperature, in the presence of a catalyst such as DMAP. Alternatively, commercially available N xcex1-acyl-aminoacid residues may be employed.
The resulting products may be purified by crystallization from suitable solvents or, if necessary, by known chromatographic techniques such as reversed-phase chromatography and ion-exchange chromatography.
Hereinbelow, examples of preparation of some modified oligopeptides according to the invention are provided.
HPLC analysis of the aminoacid derivatives, of the protected fragments and of the modified oligopeptides were carried out at the following experimental conditions:
Column: Lichrosorb RP-18;
Temperature: 25xc2x0 C. (unless otherwise mentioned)
Flow: 1.5 ml/min
Detector: Jasco 875-UV (230nm)
Eluent A: 90% water, 10% acetonitrile, 0.1% trifluoroacetic acid. (TFA)
Eluent B: acetonitrile, 0.1% TFA
Eluent C: water, 0.1% TFA
Gradients: (I): from 0 to 40% B in A (20xe2x80x2), to 80% B in A (10xe2x80x2)
(II): from 0 to 50% A in C (20xe2x80x2), to 100% A
(3xe2x80x2), to 40% B in A 20xe2x80x2.
Unless otherwise mentioned, all the synthetic steps are carried out at room temperature.
The composition and the ratio of aminoacids were determined after hydrolysis with HCl 6M at 110xc2x0 C. for 22 hours, by a Beckman SYSTEM GOLD aminoacid analyzer.
To be more clear, the meanings of the abbreviations employed in the following examples are listed hereinbelow:
BDHA-Cl=benzyldimethylhexadecylammonium chloride
Boc=tert.-butyloxycarbonyl
(Boc)2O=di-tert.butyl-dicarbonate
BOP=benzotriazol-1-yl-oxy-tris-(dimethylammino)-phosphonium hexafluorophosphate
BSA=N,O-bis(trimethylsilyl)-acetamide
DCC=dicyclohexylcarbodiimide
DMAP=4-dimethylamino-pyridina
DMF=dimethylformamide
F=fluoride
HOBT=1-hydroxy-benzotriazole
Pmc=2,2,5,7,8-pentamethyl-chroman-6-sulfonyl
PyBrop=bromo-tris-pirrolidinium-phosphonium hexafluorophosphate
tBu=tert.-butyl
TBAF=tetrabutylammonium fluoride
TEA=triethylamine
TFA=trifluoroacetic acid
Su=succinimide
Z=benzyloxycarbonyl